摘要
运用RT-PCR技术从由刀豆蛋白(ConA)诱导培养的荣昌猪外周血淋巴细胞(PBMC)扩增出猪白细胞介素15(pIL-15)完整开放阅读框(ORF),共489 bp,编码162 aa。与已公布的2个猪IL-15基因核苷酸同源性均为99.4%。分子进化分析表明其与人及哺乳动物IL-15基因的进化关系较近,而与鸡的IL-15基因进化距离较远。运用PCR技术从含荣昌猪IL-15开放阅读框序列质粒中扩增其成熟蛋白编码基因,共345 bp。将其定向克隆于原核表达载体pET-32a(+)后在E.coliBL21中诱导表达,SDS-PAGE结果显示表达的融合蛋白约为34 ku,重组蛋白以包涵体形式表达,表达产物约占菌体总蛋白的38.7%;Western blot分析表明,在相对分子质量34 ku处有一条特异性带;对诱导重组菌进行转录检测得到约356 bp的特异性片段。MTT法试验证实,表达产物初步纯化、透析复性后,可明显增强淋巴细胞的增殖。荣昌猪IL-15成熟肽成功在体外表达,获得的高效表达的产物具有一定的生物学活性。
The complete open reading frame （ORF）of interleukin-15 gene was amplified from Rongchang porcine peripheral blood lymphocyte stimulated by ConA, which includes 489bp and encodes 162aa. Sequence analysis indicated that the nucleotide shared 99.4% homology with two other porcine IL-15 genes reported previously. Phylogenetic analysis showed that the evolution relationship is comparatively close to the human and mammal IL-15 gene while it is fairly away from poultry. The gene encoding mature IL-15 protein was amplified by PCR from the recombinant plasmid containing IL-15, which includes 345 bp in all. Then it was cloned directionaly into prokaryotic expression vector pET-32a （＋） and the fusion expression was induced in E. coli BL21. SDS-PAGE demonstrated that the fusion protein expressed in form of inclusion body is approximately 34 ku, the product of expression account 38. 7% of the total bacterium proteins. Western-blotting analysis indicated that there is a specific electrophoresis strip at the place where the relative molecular mass is about 34 ku. A 356 bp specific fragment strip was observed by detection after the recombinant bacteria was transcribed. After the protein was purified roughly and renatured, MTT essay confirmed that it can enhance lymphocyte proliferation obviously. Rongchang porcine interleukinl5 mature peptide was expressed successfully in vitro and it possesses certain biological activities.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2007年第12期1351-1356,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家"973"项目(2004CCA011800)
教育部长江学者和创新团队发展计划资助(IRT0555)
